The present invention relates to a novel haloperoxidase whose activity is substantially uninhibited by relatively high concentrations of H.sub.2 O.sub.2 and hypohalous acid, and to a reaction method using such enzyme.
There has been considerable interest in recent years in enzymatic halogenation reactions. U.S. Pat. Nos. 4,247,641 and 4,284,723 describe the use of haloperoxidase enzymes to produce epoxides from alkenes. Other recent research activity in this field has explored the use of haloperoxidase enzymes to produce halogenated ketones from alkynes, alpha, gamma-halohydrins from cyclopropanes, and dihalogenated products from alkenes and alkynes.
Haloperoxidase enzymes known in the prior art which have been used in such studies include chloroperoxidase derived from the fungus Caldariomyces fumago, bromoperoxidase from algae, lactoperoxidase from milk, thyroid peroxidase from the thyroid, myeloperoxidase from leukocytes, and horseradish peroxidase from horseradish. These enzymes are described generally in Morrison, et al, Ann. Rev. Biochem., 45, 861 (1976). Each of these known haloperoxidases is a heme-containing protein.
The term haloperoxidase is used herein to include chloroperoxidases, bromoperoxidases and iodoperoxidases enzymes. A chloroperoxidase, as that term is used herein, is an enzyme capable of oxidizing chloride, bromide, or iodide ions to the corresponding hypohalous acid, with the consumption of H.sub.2 O.sub.2. A bromoperoxidase can oxidise iodide and bromide, but not chloride ions, and an iodoperoxidase can oxidize iodide ions only, both of the latter enzymes requiring H.sub.2 O.sub.2 as a substrate. The hypohalous acid which is formed in a haloperoxidase reaction may react with, and halogenate, a suitable acceptor compound. Hypohalous acid may also react with halide in solution to form the corresponding halogen. The latter reaction is favored where the ratio of hypohalous acid to acceptor compound is high, and also where the solution halide is iodide.
In a haloperoxidase-catalysed halogenation reaction, both substrate H.sub.2 O.sub.2 and hypohalous acid intermediate are reactive toward heme-containing haloperoxidases, and either can produce significant enzyme inactivation at elevated concentrations. It is likely that such inactivation involves a change in the oxidation state of the enzyme heme group. Enzyme inactivation by H.sub.2 O.sub.2 can be minimized by performing the halogenation reaction at low H.sub.2 O.sub.2 concentrations, preferably under conditions where H.sub.2 O.sub.2 is generated at a low steady state level during reaction. Likewise, enzyme inactivation by hypohalous acid can be controlled by limiting the build-up of hypohalous acid in the reaction, particularly by maintaining a relatively high concentration of acceptor compound in the reaction medium. It can be appreciated that the reaction conditions necessary to minimize enzyme inactivation by H.sub.2 O.sub.2 or hypohalous acid may impose serious rate limitations on the reaction and mandate careful monitoring of the reaction components during the course of the reaction. For these reasons, the usefulness of heme-containing haloperoxidases in industrial halogenation reaction applications has been limited.
Accordingly, it is an important object of the present invention to provide a method for producing a haloperoxidase which is substantially more resistant to inactivation by H.sub.2 O.sub.2 and hypohalous acid than known heme-containing haloperoxidases.
A more specific object of the invention is to provide a method for producing a haloperoxidase whose activity is substantially resistant to incubation at room temperature in up to 0.3M H.sub.2 O.sub.2 for up to 25 hours, and up to 0.5 mM HOCl for up to two minutes.
Providing a method for producing a stable haloperoxidase which is readily obtained by fermentation, easily isolated, has a pH optimum between about 4 and 9, and can oxidize chloride ions is still another object of the invention.
Yet another object of the invention to provide a method of using a stable haloperoxidase to oxidize halide ions in the presence of H.sub.2 O.sub.2.
The invention contemplates a method for producing a haloperoxidase which is substantially resistant to levels of H.sub.2 O.sub.2 and hypohalous acid which are known to produce rapid inactivation of heme-containing haloperoxidase enzymes known in the prior art. Two preferred fungal sources of the haloperoxidase are Curvularia inaequalis, NRRL Deposit No. 15147, and Drechslera sp., NRRL Deposit No. 15146. The stable haloperoxidase may be isolated readily by preparing a filtrate of fungal growth material and collecting the enzyme material which precipitates at a selected ammonium sulfate concentration.
The invention further contemplates a method of oxidizing a halide to the corresponding hypohalous acid in an enzymatic reaction which can be performed without appreciable enzyme inactivation, at H.sub.2 O.sub.2 and hypohalous acid concentrations which produce rapid inactivation of known heme-containing haloperoxidases. The reaction may contain a suitable acceptor compound which is to be halogenated.
These and other objects and features of the present invention will become more fully apparent from the following detailed description of the invention.